Project 2 Autonomic control of cardiovascular function depends on a highly coordinated interaction between vagal cholinergic innervation and noradrenergic sympathetic projections. In the prior project period, the Blakely project evaluated the contribution of altered noradrenergic signaling via genetic modulation of norepineprhine transporters (NETs). In the current project period, focus shifts to understanding contributions to cardiovascular phenotypes in mouse and man contributed by genetic alterations in cholinergic signaling. In particular, attention is focused on the presynaptic, high-affinity choline transporter (CHT) that pharmacological and genetic studies indicate is required to sustain cholinergic signaling through presynaptic, high-affinity choline uptake (HACU) in cholinergic terminals. The Blakely lab first cloned mouse and human CHT and has developed CHT-specific antibodies and CHT-deficient mouse models to exploit in the current project. Blakely's group examines the hypothesis that CHT is a highly regulated component off cholinergic signaling in the heart, owing to both activity-dependent shuttling of CHT proteins to the plasma membrane and transporter catalytic activation. Secondly, they examine how genetically altered CHT expression impacts parasympathetic ACh production and release and ultimately cardiovascular function. In Specific Aim 1, they establish the localization and activity of CHT in the normal mouse heart, including evaluation of subcellular distribution, transport properties and activity dependent-regulation, and CHT support for ACh synthesis and release.. Studies include light and EM immunocytochemical studies of CHT distribution in the mouse heart as well as functional and subcellular fractionation studies on atrial synaptosomes. In Specific Aim II, they determine the impact of genetic loss of CHT on cardiac HACU, ACh levels and ACh/NE receptor expression/sensitivity. Studies include CHT transport and biotinylation studies as well as HPLC and radioligand measures of ACh/NE signaling potential, comparing wildtype to CHT heterozygous mice. In Specific Aim 3, they elucidate the physiological impact of genetic variation/disruption in CHT. Studies include telemeterized recordings of cardiac function in awake, unanesthetized wildtype and CHT heterozygous mice as well as evaluations of the frequency and cardiovascular impact of human CHT polymorphisms.